Volume 4 • 2016
10.1093/conphys/cow027
Research article
Maximal oxygen consumption increases with temperature in the European eel (Anguilla anguilla) through increased heart rate and arteriovenous extraction Débora Claësson, Tobias Wang and Hans Malte* Department of Bioscience, Zoophysiology, Aarhus University, C. F. Møllers Allé 3, DK08000 Aarhus C, Denmark *Corresponding author: Department of Bioscience, Zoophysiology, Aarhus University, C. F. Møllers Allé 3, DK08000 Aarhus C, Denmark. Tel: +45 8715 5994. Email: [email protected] ..............................................................................................................................................................
Global warming results in increasing water temperature, which may represent a threat to aquatic ectotherms. The rising temperature affects ecology through physiology, by exerting a direct limiting effect on the individual. The mechanism controlling individual thermal tolerance is still elusive, but some evidence shows that the heart plays a central role, and that insufficient transport of oxygen to the respiring tissues may determine the thermal tolerance of animals. In this study, the influence of the heart in thermal limitation was investigated by measurements of aerobic scope in the European eel (Anguilla anguilla) together with measurements of cardiac output during rest and activity. Aerobic capacity was not limited by an acutely increased temperature in the European eel. Oxygen demand was met by an increase in heart rate and arteriovenous extraction. These findings suggest that thermal tolerance during exposure to acute temperature changes is not defined by oxygen transport capacity in the eel, and other mechanisms may play a central role in limiting thermal tolerance in these fish. Key words: Aerobic scope, blood flow, heart rate, oxygen- and capacity-limited thermal tolerance, oxygen consumption, temperature Editor: Steven Cooke Received 22 December 2015; Revised 27 May 2016; accepted 5 June 2016 Cite as: Claësson D, Wang T, Malte H (2016) Maximal oxygen consumption increases with temperature in the European eel (Anguilla anguilla) through increased heart rate and arteriovenous extraction. Conserv Physiol 4(1): cow027; doi:10.1093/conphys/cow027. ..............................................................................................................................................................
Introduction The current shifts in phenology, distribution and abundance of aquatic ectotherms have been correlated with direct effects of rising temperatures on bodily functions, and future conservation strategies, therefore, depend on an ability to understand how temperature affects physiological processes at the organism level (Pörtner, 2012; Schulte, 2015). In fishes and other ectotherms, elevated temperature increases the
standard metabolic rate (SMR), measured as the minimal oxygen consumption (Ṁ O2 ) of inactive and post-absorptive animals that are not recovering from anaerobic exercise (Fry and Hart, 1948). The difference between the maximal oxygen consumption (Ṁ O2 max ) and SMR is defined as the aerobic scope (AS; Fry and Hart, 1948), and this capacity is used extensively to assess potential impacts of climate change on fishes (Pörtner and Knust, 2007; Wang and Overgaard, 2007; Pörtner and Farrell, 2008).
.............................................................................................................................................................. The Author 2016. Published by Oxford University Press and the Society for Experimental Biology. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Research article Conservation Physiology • Volume 4 2016 ..............................................................................................................................................................
The so-called ‘oxygen- and capacity-limited thermal tolerance (OCLTT) model’ states that the failure of oxygen transport systems to match bodily oxygen demand dictates thermal tolerance (Pörtner and Knust, 2007; Pörtner and Farrell, 2008). This model predicts that performance quickly deteriorates above the optimal temperature for AS (ToptAS) as a result of the inability of the oxygen transport systems to cope with the higher oxygen demand (Frederich and Pörtner, 2000; Pörtner, 2010). As a non-exclusive alternative, the temperature tolerances of physiological and biochemical capacities have co-evolved, so that the ToptAS coincides with the temperature at which performance (such as locomotion and growth) and fitness (i.e. survival and reproductive success) are optimal (e.g. Clark et al., 2013). In this case, limited oxygen delivery is not the mechanistic cause for the critical thermal maximum (CTmax). Any component of the oxygen transport cascade responsible for bringing oxygen from the water to the mitochondria may limit Ṁ O2 max , but given that arterial oxygen levels normally remain high, most studies emphasize the convective transport of oxygen in the blood as a limitation (Kiceniuk and Jones, 1977; Wang and Malte, 2011). Cardiac output (Q)̇ can be increased through elevations of stroke volume (Vs) and/or heart rate (fH), both of which depend on adequate oxygen supply to the cardiac muscle. In fishes, the myocardial oxygen delivery may be limited at high temperatures, because the spongy myocardium is devoid of coronary perfusion; hence, it depends entirely on oxygen availability in the oxygen-poor, venous blood. We hypothesize that AS decreases above ToptAS in concert with a gradual collapse in Q.̇ To investigate this, Ṁ O2 and Ṁ O2 max were measured over a broad temperature range in European eels (Anguilla anguilla). The eel provides a good model for investigating cardiac variables, owing to their easily exposable ventral aorta. With a flow probe placed around the ventral aorta, Q̇ and fH were quantified at increasing temperatures. Thus, the question of whether there was a collapse in the cardiac function in European eels with the increasing temperatures could be addressed.
Transponder (PIT tag from Loligo Systems, Tjele, Denmark) inserted through a 5 mm incision in the ventral body wall under immersion anaesthesia (0.5 g/l benzocaine). All experiments were approved by the Danish Animal Experiments Inspectorate (permit no. 2012-15-2934-00246).
Determination of the critical thermal maximum The CTmax was estimated as the temperature at which the eels began to lose equilibrium. Eight eels (311 ± 19 g) were placed in a 135 litre container filled with aerated freshwater at 18°C. After 1 h, the temperature was increased at 1.8°C h−1 using a Julabo FP51 cooler. When the eel was unable to maintain equilibrium, temperature was registered, and it was immediately transferred to fully aerated water for recovery at 18°C.
Measurements of standard metabolic rate and maximal oxygen uptake The Ṁ O2 was measured in 41 eels (324 ± 10 g) immediately after enforced activity and subsequent rest using intermittent closed respirometry (Steffensen, 1989). This method and protocol provides robust measures of maximal metabolic (aerobic) rate (MMR) and SMR in inactive and resting fish species (Clark et al., 2013). A 135 litre tank was filled with aerated freshwater and connected to a Heto HMT 200 thermostat to maintain temperatures within ±0.5°C. The fish were enclosed in submerged respirometers (2.5 litres) where a galvanic oxygen electrode (Oxyguard mini connected to a Loligo Systems Loli-DAQ data acquisition box), calibrated daily in anoxic and fully aerated water, measured the decline in water PO2 at 1 Hz. Water constantly circulated past the electrode at a steady flow, so Ṁ O2 could be calculated from the slope of linear regression of PO2 vs. time (in kilopascals per minute) using the following equation:
Ṁ O2 =
βO2 V Mb
×
ΔPO2 , Δt
Experimental animals
where ΔPO2 / Δt is the change in water oxygen pressure per unit time, β is the oxygen solubility in water, V is the volume of the respirometer, and Mb is the body mass of the fish.
European eels (A. anguilla) of undetermined sex (299 ± 84 g) were purchased from Lyksvad fish farm (Vamdrup, Denmark) and kept at Aarhus University for no less than 3 weeks in normoxic [partial pressure of oxygen (PO2 ) >140 mmHg] and non-chlorinated tap water at 18°C, with a photoperiod of 12 h light–12 h dark. The water was recirculated and biologically filtered (Akva Group, Vejle, Denmark) at a flow of 1000 l/h, and the temperature and oxygen were monitored continuously. The eels were fed ~0.7% body mass/day with Dan-Ex eel pellets (Biomar A/S, Brande, Denmark), but fasted for at least 48 h before experiments. All eels were tagged with FDX-B Passive Integrated
At each test temperature, the duration of the closed periods was adjusted differently to ensure that PO2 never fell below 18 kPa. The tank was connected to an ultraviolet filter to reduce bacterial growth. Throughout the subsequent 48 h, the tank was shielded to minimize visual disturbance during measurements of SMR. The system was automated, and after each measurement the respirometers were flushed for 200 s to replenish O2 and get rid of CO2 and other excretion products. The order of test temperatures and the eel used were randomized to minimize time bias. At the end of each experiment, bacterial Ṁ O2 was measured in the empty respirometers for 1 h, and the value, which never exceeded 10% of
Materials and methods
..............................................................................................................................................................
2
Research article Conservation Physiology • Volume 4 2016 ..............................................................................................................................................................
the fish, was subtracted. The respirometer and all tubing were carefully cleaned before the next experiment. Data were analysed using a Mathematica script (version 5.2; Wolfram Research, Champaign, IL, USA). Eels were quickly transported from the holding tank to the laboratory in water from the holding tank. Before introducing the eels to the respirometers, they were transferred to a container with water at the experimental temperature and exercised by chasing. The container was ellipsoidal and allowed for burst-and-glide swimming. One eel was transferred to the container at a time and left for 10 min before the chasing was commenced. Chasing was continued until the eels no longer responded to tactile stimuli and appeared exhausted. This procedure is suitable for an ambush predator, such as the eel, that does not undertake long periods of swimming in their freshwater cycle (Schultz et al., 2009). As a consequence, the critical swimming speed (Ucrit) protocol (Brett, 1964) would be unlikely to elicit MMR in eels (Peake and Farrell, 2006). After exhaustion, the eels were quickly returned to the respirometers, and the measurement was started immediately. The Ṁ O2 max was considered to be the highest Ṁ O2 measurement, which normally occurred during the first measurement of O2 uptake after chasing. The SMR was estimated as the mean of the 10% lowest Ṁ O2 values excluding outliers (>2 SD from the mean).
Measurements of the cardiovascular responses to exercise at various temperatures Eels were anaesthetized by immersion in freshwater containing benzocaine (0.5 g/l) until ventilation ceased. Next, the eels were placed on an operating table, where their gills could be irrigated with aerated freshwater containing benzocaine (0.1 g/l). Xylocaine (0.3 ml, 20 mg/ml) was injected subcutaneously before a 1 cm ventral mid-line incision allowed a Transonic flowprobe to be placed around the ventral aorta. The incision was closed with three sutures, and the probe lead was fixed ventrally on the skin with stitches. For recovery, the eels were placed in individual 10 litre restrainers contained in a 120 litre aquarium with aerated freshwater at 18°C. The procedure took
10.1093/conphys/cow027
Research article
Maximal oxygen consumption increases with temperature in the European eel (Anguilla anguilla) through increased heart rate and arteriovenous extraction Débora Claësson, Tobias Wang and Hans Malte* Department of Bioscience, Zoophysiology, Aarhus University, C. F. Møllers Allé 3, DK08000 Aarhus C, Denmark *Corresponding author: Department of Bioscience, Zoophysiology, Aarhus University, C. F. Møllers Allé 3, DK08000 Aarhus C, Denmark. Tel: +45 8715 5994. Email: [email protected] ..............................................................................................................................................................
Global warming results in increasing water temperature, which may represent a threat to aquatic ectotherms. The rising temperature affects ecology through physiology, by exerting a direct limiting effect on the individual. The mechanism controlling individual thermal tolerance is still elusive, but some evidence shows that the heart plays a central role, and that insufficient transport of oxygen to the respiring tissues may determine the thermal tolerance of animals. In this study, the influence of the heart in thermal limitation was investigated by measurements of aerobic scope in the European eel (Anguilla anguilla) together with measurements of cardiac output during rest and activity. Aerobic capacity was not limited by an acutely increased temperature in the European eel. Oxygen demand was met by an increase in heart rate and arteriovenous extraction. These findings suggest that thermal tolerance during exposure to acute temperature changes is not defined by oxygen transport capacity in the eel, and other mechanisms may play a central role in limiting thermal tolerance in these fish. Key words: Aerobic scope, blood flow, heart rate, oxygen- and capacity-limited thermal tolerance, oxygen consumption, temperature Editor: Steven Cooke Received 22 December 2015; Revised 27 May 2016; accepted 5 June 2016 Cite as: Claësson D, Wang T, Malte H (2016) Maximal oxygen consumption increases with temperature in the European eel (Anguilla anguilla) through increased heart rate and arteriovenous extraction. Conserv Physiol 4(1): cow027; doi:10.1093/conphys/cow027. ..............................................................................................................................................................
Introduction The current shifts in phenology, distribution and abundance of aquatic ectotherms have been correlated with direct effects of rising temperatures on bodily functions, and future conservation strategies, therefore, depend on an ability to understand how temperature affects physiological processes at the organism level (Pörtner, 2012; Schulte, 2015). In fishes and other ectotherms, elevated temperature increases the
standard metabolic rate (SMR), measured as the minimal oxygen consumption (Ṁ O2 ) of inactive and post-absorptive animals that are not recovering from anaerobic exercise (Fry and Hart, 1948). The difference between the maximal oxygen consumption (Ṁ O2 max ) and SMR is defined as the aerobic scope (AS; Fry and Hart, 1948), and this capacity is used extensively to assess potential impacts of climate change on fishes (Pörtner and Knust, 2007; Wang and Overgaard, 2007; Pörtner and Farrell, 2008).
.............................................................................................................................................................. The Author 2016. Published by Oxford University Press and the Society for Experimental Biology. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Research article Conservation Physiology • Volume 4 2016 ..............................................................................................................................................................
The so-called ‘oxygen- and capacity-limited thermal tolerance (OCLTT) model’ states that the failure of oxygen transport systems to match bodily oxygen demand dictates thermal tolerance (Pörtner and Knust, 2007; Pörtner and Farrell, 2008). This model predicts that performance quickly deteriorates above the optimal temperature for AS (ToptAS) as a result of the inability of the oxygen transport systems to cope with the higher oxygen demand (Frederich and Pörtner, 2000; Pörtner, 2010). As a non-exclusive alternative, the temperature tolerances of physiological and biochemical capacities have co-evolved, so that the ToptAS coincides with the temperature at which performance (such as locomotion and growth) and fitness (i.e. survival and reproductive success) are optimal (e.g. Clark et al., 2013). In this case, limited oxygen delivery is not the mechanistic cause for the critical thermal maximum (CTmax). Any component of the oxygen transport cascade responsible for bringing oxygen from the water to the mitochondria may limit Ṁ O2 max , but given that arterial oxygen levels normally remain high, most studies emphasize the convective transport of oxygen in the blood as a limitation (Kiceniuk and Jones, 1977; Wang and Malte, 2011). Cardiac output (Q)̇ can be increased through elevations of stroke volume (Vs) and/or heart rate (fH), both of which depend on adequate oxygen supply to the cardiac muscle. In fishes, the myocardial oxygen delivery may be limited at high temperatures, because the spongy myocardium is devoid of coronary perfusion; hence, it depends entirely on oxygen availability in the oxygen-poor, venous blood. We hypothesize that AS decreases above ToptAS in concert with a gradual collapse in Q.̇ To investigate this, Ṁ O2 and Ṁ O2 max were measured over a broad temperature range in European eels (Anguilla anguilla). The eel provides a good model for investigating cardiac variables, owing to their easily exposable ventral aorta. With a flow probe placed around the ventral aorta, Q̇ and fH were quantified at increasing temperatures. Thus, the question of whether there was a collapse in the cardiac function in European eels with the increasing temperatures could be addressed.
Transponder (PIT tag from Loligo Systems, Tjele, Denmark) inserted through a 5 mm incision in the ventral body wall under immersion anaesthesia (0.5 g/l benzocaine). All experiments were approved by the Danish Animal Experiments Inspectorate (permit no. 2012-15-2934-00246).
Determination of the critical thermal maximum The CTmax was estimated as the temperature at which the eels began to lose equilibrium. Eight eels (311 ± 19 g) were placed in a 135 litre container filled with aerated freshwater at 18°C. After 1 h, the temperature was increased at 1.8°C h−1 using a Julabo FP51 cooler. When the eel was unable to maintain equilibrium, temperature was registered, and it was immediately transferred to fully aerated water for recovery at 18°C.
Measurements of standard metabolic rate and maximal oxygen uptake The Ṁ O2 was measured in 41 eels (324 ± 10 g) immediately after enforced activity and subsequent rest using intermittent closed respirometry (Steffensen, 1989). This method and protocol provides robust measures of maximal metabolic (aerobic) rate (MMR) and SMR in inactive and resting fish species (Clark et al., 2013). A 135 litre tank was filled with aerated freshwater and connected to a Heto HMT 200 thermostat to maintain temperatures within ±0.5°C. The fish were enclosed in submerged respirometers (2.5 litres) where a galvanic oxygen electrode (Oxyguard mini connected to a Loligo Systems Loli-DAQ data acquisition box), calibrated daily in anoxic and fully aerated water, measured the decline in water PO2 at 1 Hz. Water constantly circulated past the electrode at a steady flow, so Ṁ O2 could be calculated from the slope of linear regression of PO2 vs. time (in kilopascals per minute) using the following equation:
Ṁ O2 =
βO2 V Mb
×
ΔPO2 , Δt
Experimental animals
where ΔPO2 / Δt is the change in water oxygen pressure per unit time, β is the oxygen solubility in water, V is the volume of the respirometer, and Mb is the body mass of the fish.
European eels (A. anguilla) of undetermined sex (299 ± 84 g) were purchased from Lyksvad fish farm (Vamdrup, Denmark) and kept at Aarhus University for no less than 3 weeks in normoxic [partial pressure of oxygen (PO2 ) >140 mmHg] and non-chlorinated tap water at 18°C, with a photoperiod of 12 h light–12 h dark. The water was recirculated and biologically filtered (Akva Group, Vejle, Denmark) at a flow of 1000 l/h, and the temperature and oxygen were monitored continuously. The eels were fed ~0.7% body mass/day with Dan-Ex eel pellets (Biomar A/S, Brande, Denmark), but fasted for at least 48 h before experiments. All eels were tagged with FDX-B Passive Integrated
At each test temperature, the duration of the closed periods was adjusted differently to ensure that PO2 never fell below 18 kPa. The tank was connected to an ultraviolet filter to reduce bacterial growth. Throughout the subsequent 48 h, the tank was shielded to minimize visual disturbance during measurements of SMR. The system was automated, and after each measurement the respirometers were flushed for 200 s to replenish O2 and get rid of CO2 and other excretion products. The order of test temperatures and the eel used were randomized to minimize time bias. At the end of each experiment, bacterial Ṁ O2 was measured in the empty respirometers for 1 h, and the value, which never exceeded 10% of
Materials and methods
..............................................................................................................................................................
2
Research article Conservation Physiology • Volume 4 2016 ..............................................................................................................................................................
the fish, was subtracted. The respirometer and all tubing were carefully cleaned before the next experiment. Data were analysed using a Mathematica script (version 5.2; Wolfram Research, Champaign, IL, USA). Eels were quickly transported from the holding tank to the laboratory in water from the holding tank. Before introducing the eels to the respirometers, they were transferred to a container with water at the experimental temperature and exercised by chasing. The container was ellipsoidal and allowed for burst-and-glide swimming. One eel was transferred to the container at a time and left for 10 min before the chasing was commenced. Chasing was continued until the eels no longer responded to tactile stimuli and appeared exhausted. This procedure is suitable for an ambush predator, such as the eel, that does not undertake long periods of swimming in their freshwater cycle (Schultz et al., 2009). As a consequence, the critical swimming speed (Ucrit) protocol (Brett, 1964) would be unlikely to elicit MMR in eels (Peake and Farrell, 2006). After exhaustion, the eels were quickly returned to the respirometers, and the measurement was started immediately. The Ṁ O2 max was considered to be the highest Ṁ O2 measurement, which normally occurred during the first measurement of O2 uptake after chasing. The SMR was estimated as the mean of the 10% lowest Ṁ O2 values excluding outliers (>2 SD from the mean).
Measurements of the cardiovascular responses to exercise at various temperatures Eels were anaesthetized by immersion in freshwater containing benzocaine (0.5 g/l) until ventilation ceased. Next, the eels were placed on an operating table, where their gills could be irrigated with aerated freshwater containing benzocaine (0.1 g/l). Xylocaine (0.3 ml, 20 mg/ml) was injected subcutaneously before a 1 cm ventral mid-line incision allowed a Transonic flowprobe to be placed around the ventral aorta. The incision was closed with three sutures, and the probe lead was fixed ventrally on the skin with stitches. For recovery, the eels were placed in individual 10 litre restrainers contained in a 120 litre aquarium with aerated freshwater at 18°C. The procedure took
